FcγRIIb inhibition promotes phagocytosis of rituximab-treated CLL cells. (A) Phagocytosis assays: CFSE-labeled CLL cells were treated with GA101_gly for 6 hours and then added to normal human macrophages for phagocytosis. Representative images are shown by confocal microscopy (left) and after May-Grünwald-Giemsa staining by bright field microscopy (right). Bar represents 10 μm. A Leica TCS-SP5 laser scanning confocal miscroscope (10× eye piece, 40× objective oil immersion lens) and Olympus CKX21 inverted microscope with 10×/0.25PH lens with 40× objective oil immersion lens was used, respectively. (B) The assay in panel A was repeated but CFSE-labeled CLL cells were treated with either GA101_gly or rituximab for 15 minutes or 6 hours before adding to the macrophages. Anti–CD16-APC was used to identify the macrophages, and results were analyzed by flow cytometry. Representative dot plots are shown for untreated and treated cells (top panels). Double-positive cells include both macrophages that have phagocytosed CFSE-labeled CLL cells, and also macrophages that have them rosetted on their surface. However, visual analysis of confocal images as in panel A suggests that > 90% of the CFSE-labeled CLL cells have been phagocytosed. The graph (bottom panel) shows the reduction in the % double positive cells between 15 minutes and 6 hours. Each point shows the mean of triplicate samples, and 3 different CLL patients are shown. Different symbols are used to identify samples from the same patients. (C) As in panel B, but CLL cells were treated with rituximab only, or in combination with AT10 F(ab′)₂ for 15 minutes or 6 hours and phagocytosis measured. Triplicate samples are shown; the experiment has been repeated twice.